The long-term goal of the proposed research is to understand how cells preserve genomic integrity. Genetic instability is one characteristic of cancer cells and explains how they accumulate multiple genetic alterations that promote tumorigenic growth. Cells with defective DNA damage and replication stress response capabilities exhibit high rates of genomic instability. Therefore, we aim to define the components of DNA damage/replication stress response pathways and determine how they work cooperatively to prevent cancer by regulating the cell cycle, promoting DNA repair or initiating apoptosis. The ATR (ATM and rad3-related) kinase functions at the apex of a DNA damage and replication stress response pathway. In the previous funding period, we defined several mechanisms by which the ATR-interacting protein (ATRIP) promotes ATR specific responses to genotoxic stress. Our results as well as those of other researchers suggest a multi-step model for ATR signaling regulation, but many questions remain unanswered. We hypothesize that ATR-ATRIP activation in response to genotoxic stress is regulated through multiple mechanisms including RPA-dependent localization, TopBP1 binding, and phosphorylation. This hypothesis will be tested using a combination of biochemical and genetic approaches. Components of the ATR signaling pathway are frequently mutated in several human diseases; ATR activation is a major cellular response to most commonly used cancer therapies; and the ATR pathway is expected to be a useful drug target. Therefore, these mechanistic studies focus on highly significant biological questions.